Biliverdin IX-alpha (biliverdin IXα) is the most common form of several biliverdin isomers found in nature (FIG. 1C). It is produced from heme (also named iron protoporphyrin IX) (FIG. 1A) by the enzyme heme oxygenase. In animals, biliverdin IXα is reduced by the enzyme biliverdin reductase to bilirubin IXα which is the major form of several bilirubin isomers (FIG. 1B). One known role of biliverdin IXα in nature is in animals as an intermediate in hemoglobin breakdown as red blood cells are degraded in phagocytes (FIG. 2). The hemoglobin prosthetic group, heme, with its bound iron, is released in this degradative process, and heme is converted by HO to biliverdin IXα. Biliverdin IXα is then converted to bilirubin IXα by the enzyme biliverdin reductase (FIG. 2). Bilirubin IXα is consecutively bound to serum albumin and then in the liver to glucoronic acid (conjugated bilirubin) which confers a relatively high degree of water solubility. Conjugated bilirubin is then excreted in the bile. Overall, this process is viewed as a process for animals (e.g. humans) to degrade and eliminate heme—which is toxic when accumulated.
Biliverdin IXα is also made by microbes. For example, biliverdin IXα is a precursor to microbial phycobilins, i.e. phycocyanobilin (pcb) and phycoerythrobilin (peb). Pcb and peb are the pigment molecules for the light-harvesting complexes of photosynthetic cyanobacteria, phycocyanin and phycoerythrin, respectively. These complexes collect light energy (for example solar energy) and funnel it to photosynthetic reaction centers where the energy is converted into chemical energy) (FIG. 3). Pcb is also the pigment for phytochrome—a light sensing receptor that occurs in plants and other cells. An analogous receptor, bacteriophytochrome, is found in certain bacteria. The pigment for bacteriophytochrome is biliverdin IXα rather than pcb, which reveals yet another biological role for biliverdin IXα. These latter bacteria—like all microbes—either do not produce or do not accumulate bilirubin IXα. The lack of bilirubin IXα accumulation by microbes is either a consequence of lacking biliverdin reductase or the conversion of bilirubin IXα to bile pigments such as those involved in photosynthetic light-harvesting.
Bilirubin IXα is known to associate with cell membranes where it quenches the propagation of reactive oxygen species (ROS). It is therefore believed to confer protection to membrane lipid and protein components against oxidative damage. Thus, an additional suggested function of biliverdin IXα is to serve as the immediate source of bilirubin IXα which in turn acts as a cytoprotective antioxidant and anti-inflammatory agent against cell damaging ROS (FIG. 4). Although bilirubin IXα (and not biliverdin IXα) is believed to be the cytoprotective antioxidant, it is observed that biliverdin IXα is more effective than bilirubin IXα when administered at tissue injury/inflammatory sites where ROS are prevalent. One explanation for the higher efficacy of biliverdin IXα is that it is more hydrophilic than bilirubin IXα and therefore has better access to tissue sites where it is then reduced by biliverdin reductase to bilirubin IXα. Another explanation is that when biliverdin IXα binds to biliverdin reductase, this enzyme is activated and initiates a cell signaling cascade that results in the production of the anti-inflammatory cytokine interferon-10.
There is increasing evidence that biliverdin IXα can be used as a cytoprotective therapeutic agent. Examples of clinical applications of biliverdin IXα include treatment of vasoconstriction (U.S. Patent Application Publication No. 20030027124); coronary artery disease (artherosclerosis); ischemia/reperfusion injuries after small intestinal, heart, and kidney transplantation; severe sepsis; injuries from liver grafts; and prevention of intimal hyperplasia induced by vascular injury. Today, biliverdin IXα is predominantly derived by chemical oxidation of bilirubin IXα or by using the enzyme bilirubin oxidase (U.S. Pat. No. 5,624,811). Bilirubin IXα is extracted from the bile of various mammals, especially from swine or other livestock. Commercial animal bilirubin IXα preparations are often contaminated with conjugated bilirubin and isomers (e.g. bilirubin XIIIα) (Reisinger et al. 2001; U.S. Pat. No. 431,166). As a result, biliverdin IXα derived from bilirubin IXα preparations using oxidative processes or enzymes may also contain isomers. The clinical consequences of using biliverdin IXα contaminated with such isomers are not clear. In addition, the use of biliverdin IXα preparations derived from animal bilirubin carries the risk of prion contamination often associated with materials derived from animal sources.
A recent claim (U.S. Pat. No. 7,504,243) for biliverdin IXα production by a yeast depends on addition of hemoglobin (from animal blood) to the growth culture as a source of heme. Another report shows biliverdin IXα synthesis by Escherichia coli expressing a heterologous HO gene of animal origin (rat). The biliverdin IXα was produced at low levels and appears to remain cell-bound.
The limited amounts of biliverdin IXα produced by yeast and E. coli expressing heterologous HO genes could result from restricted access to heme. In E. coli, the biosynthesis of heme is regulated at the initial step of tetrapyrrole biosynthesis—the synthesis of 5-aminolevulinic acid (ALA) by the C5-pathway. The C5 pathway involves conversion of glutamate to glutamyl-tRNA by glutamyl-tRNA synthetase, reduction to glutamate-γ-semialdehyde by an NADPH-dependent glutamyl tRNA reductase and transamination by glutamate-γ-semialdehyde aminomutase to form ALA. The C5 pathway is feedback-inhibited by heme and, as a consequence, the cellular levels of heme are kept low. In contrast, mammals, plants, and certain bacteria such as Rhodobacter sphaeroides produce ALA from glycine and succinyl-CoA via the enzyme ALA synthetase. This latter mechanism for ALA biosynthesis is termed the “C4 pathway.” The C4 pathway ALA synthetase is not subject to feedback inhibition by heme. It therefore allows the accumulation of heme and higher cellular concentrations. When combined with the C4 pathway for ALA synthesis, HO will have greater access to its substrate, heme, resulting in increased potential for producing biliverdin IXα.